The present invention relates generally to compositions and methods useful for the study of mitogen-activated protein kinase cascades and for treating diseases associated with such pathways. The invention is more particularly related to the mitogen-activated protein kinase p38-2, and variants thereof that stimulate phosphorylation and activation of substrates, such as ATF2. The present invention is also related to the use of such polypeptides to identify antibodies and other agents that inhibit or activate signal transduction via the p38-2 kinase cascade.
Mitogen-activated protein kinases (MAPKs) are members of conserved signal transduction pathways that activate transcription factors, translation factors and other target molecules in response to a variety of extracellular signals. MAPKs are activated by phosphorylation at a dual phosphorylation motif with the sequence Thr-X-Tyr by mitogen-activated protein kinase kinases (MAPKKs). In higher eukaryotes, the physiological role of MAPK signaling has been correlated with cellular events such as proliferation, oncogenesis, development and differentiation. Accordingly, the ability to regulate signal transduction via these pathways could lead to the development of treatments and preventive therapies for human diseases associated with MAPK signaling, such as inflammatory diseases, autoimmune diseases and cancer.
In mammalian cells, three parallel MAPK pathways have been described. The best characterized pathway leads to the activation of the extracellular-signal-regulated kinase (ERK). Less well understood are the signal transduction pathways leading to the activation of the cJun N-terminal kinase (JNK) and the p38 MAPK (for reviews, see Davis, Trends Biochem. Sci. 19:470-473 (1994); Cano and Mahadevan, Trends Biochem. Sci. 20:117-122(1995)). The identification and characterization of members of these cascades is critical for understanding the signal transduction pathways involved and for developing methods for activating or inactivating MAPKs in vivo.
Three MAPKKs capable of activating p38 in vitro have been identified. MKK3 appears to be specific for p38 (i.e., does not activate JNK or ERK), while MKK4 activates both p38 and JNK (see Derijard et al., Science 267:682-685, 1995). The third MAPKK, MEK6, appears to be a stronger and more specific in vivo stimulator of p38 phosphorylation (see U.S. patent application Ser. No. 08/576,240). These proteins appear to have utility in therapeutic methods for treating conditions associated with the p38 signal transduction pathway. However, in order to precisely tailor such therapeutic methods, and to gain an understanding of the pathways involved, it would be advantageous to identify and characterize other proteins that participate in this cascade and related MAP kinase cascades.
Accordingly, there is a need in the art for improved methods for modulating the activity of proteins involved in the MAP kinase cascades, and for treating diseases associated with such cascades. The present invention fulfills these needs and further provides other related advantages.
Briefly stated, the present invention provides compositions and methods employing the mitogen-activated protein kinase (MAPK) p38-2, or a variant thereof. In one aspect, the present invention provides polypeptides comprising the amino acid sequence provided in SEQ ID NO:2 or a variant thereof that differs only in conservative substitutions and/or modifications at no more than 25% of the amino acid residues. Such variants include constitutively active polypeptides. In a related aspect, polypeptides comprising the amino acid sequence provided in SEQ ID NO:2 modified at no more than 25% of the amino acid residues, such that the polypeptides are rendered constitutively inactive, are provided.
In other aspects, isolated DNA molecules encoding polypeptides as described above, as well as recombinant expression vectors comprising such DNA molecules and host cells transformed or tansfected with such expression vectors, are provided.
In another aspect, the present invention provides methods for phosphorylating a substrate of p38-2, comprising contacting a polypeptide as described above with a substrate of p38-2, thereby phosphorylating the substrate of p38-2. In a related aspect, methods are provided for activating a substrate of p38-2 in a patient, comprising administering to a patient a polypeptide as described above in combination with a pharmaceutically acceptable carrier, thereby activating a substrate of p38-2.
In further aspects, the present invention provides methods for screening for an agent that modulates signal transduction via the p38-2 cascade. In one embodiment the method comprises: (a) contacting a candidate agent with a polypeptide as described above, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the polypeptide to interact; and (b) subsequently measuring the ability of the candidate agent to modulate p38-2 activity, and thereby evaluating the ability of the candidate agent to modulate signal transduction via the p38 cascade. In another embodiment, the method comprises: (a) contacting a candidate agent with a polynucleotide encoding a polypeptide as described above, wherein the step of contacting is carried out under conditions and for a time sufficient to allow generation of the polypeptide and interaction between the polypeptide and the candidate agent; and (b) subsequently measuring the ability of the candidate agent to modulate p38-2 activity, and thereby evaluating the ability of the candidate agent to modulate signal transduction via the p38-2 cascade.
In yet another aspect, the present invention provides antibodies that bind to a polypeptide as described above.
In further aspects, methods are provided for treating a disorder associated with the p38-2 cascade, comprising administering to a patient a therapeutically effective amount of an agent that modulates signal transduction via the p38-2 cascade. In related aspects, methods are provided for treating a patient afflicted with a disease associated with the p38-2 cascade, comprising administering to a patient an agent that inhibits p38-2 kinase activity or that inhibits phosphorylation of p38-2.
In still further aspects, the present invention provides methods and kits for detecting mitogen activated protein kinase kinase activity in a sample. The methods comprise evaluating the ability of the sample to phosphorylate a polypeptide as described above, thereby detecting mitogen activated protein kinase kinase activity in the sample. Kits comprise p38-2 in combination with a suitable buffer.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.